Abiotic stresses, caused by too low or too high temperatures, by lack of water or by high concentrations of salts and heavy metals in soils, are responsible for great losses in agriculture, drastically reducing the production and generating losses that may exceed 50% (Boyer, 1982; Wang et al, 2003). In order to survive in such hostile environments, plants have developed a number of complex strategies that involve morphologic, physiologic and molecular alterations, in an attempt to become more tolerant (Nakashima et al., 2009; Verslues et al., 2000). Among abiotic stresses, drought has received growing attention, since, besides limiting the productivity, it has an important role in determining the distribution of the species in different ecosystems. The concern about the potential impact that climatic changes may cause on temperature and on the pluviometric patterns leads to the growing need to increase agricultural productivity (Ramalho et al, 2009). All over the world, about 70% of water available for consumption is used in agriculture and, although the irrigation is a strategy used to minimize the damages caused by lack of water, the irrigation system also has disadvantages such as the increase in the production cost and salinization of soils (Somerville & Briscoe, 2001). The classic improvement of agronomically important (e.g. Coffea arabica) with a view to develop such characteristics as flowering, higher productivity and greater resistance to pests and abiotic stresses (e.g. frosts and droughts), in spite of being successful, is also considered a slow development, which requires a huge demand for work and financial resources (Etienne et al., 2002).
Studies with transcription factors belonging to the homeobox family, more specifically to the HD-Zip family, reveal that these factors may be involved in modulating responses of plants to drought, controlling the plant development in such conditions (Deng et al., 2006; Dezar et al., 2005). The HD-Zip protein family is characterized by the presence of homeodomain (HD), coupled to an adjacent leucine zipper (LZ), which is important to the formation of homo- and heterodimers (Frank et al., 1998; Johannesson et at, 2001; Ruberti et al.; Sessa et al., 1993). The association or these two elements (HD and LZ) in a single protein is exclusive to plants, and this family represents, in number of members, from 40 to 50% of all the homeobox genes present in mosses to angiosperms. So far, four sub-DH-Zip protein subfamilies (HD-Zip I, II, III and IV) can be distinguished on the basis of the similarity of sequence and gene structure inside and outside the HD (Mukherjee et al., 2009). The expression of genes belonging specially to the HD-Zip I subfamily is generally modulated by environmental factors (e.g. light, low temperatures, salt and water stress), and its role in regulating the plant development in response to these stimuli may occasionally lead to phenotypes of greater tolerance to drought (Ariel et al., 2007).
U.S. Pat. No. 5,981,729 relates to a new gene of the homeobox family, isolated from the species Arabidopsis thatiana, which encodes the ATHB-12 transcription factor, related to the response to drought and to abscisic acid (ABA). Said gene may be cloned on expression factors to produce a recombinant DNA expression system, suitable for the transformation of plant cells and the production of transgenic plants that are more tolerant to drought. Said patent, however, does not mention or make any reference to any tolerance experiment with transgenic plants bearing said gene.
Patent WO 04/099365 describes an invention characterized by a gene isolated from Helianthus annuus, which encodes the HAHB-4 transcription factor, belonging to the DH-Zip family. The expression of the HAHB-4 gene is reduced by water deficit or by abscisic acid (ABA), and may be cloned in DNA constructs to transform host cells and plants. The transgenic plants that express the transcription factor gene are tolerant and resistant to water deficit and high salinity. In the present invention the new CAHB12 coffee gene also encodes a transcription factor of the HD-Zip family, expressed in leaves and roots of coffee plants of the species Coffea arabica cultivated in conditions of water deficit, exhibiting growing levels of expression, according to the severity of the stress experimented. Transgenic plants bearing this gene under control of the 35S promoter, which guarantees a constructive expression, that is, all the organs at high levels, exhibit greater tolerance to different conditions of water deficit, at different stages of development, as well as greater tolerance to salt stress. The levels of tolerance to salt stress of plants bearing the CAHB12 gene are even higher than those observed in plants bearing the HAHB4 gene, described in patent WO 04/099365. In the present study, besides the rate of germination in the presence of varied concentrations of NaCl (100 and 150 mM), measures of fresh weight have been evaluated, as well as the levels of lipid peroxidation mediated by water deficit monitored, through the rates of malonic aldehyde. Transgenic plants expressing the CAHB12 gene exhibited germination rages ranging from 20 to 45% higher than those of non-transgenic plants cultivated in the presence of 150 mM of NaCl. In patent WO 04/099365, the percentage of transgenic and non-transgenic plants germinated 46 hours after the beginning of the experiment was the same (100%). In the present invention, the plants belonging to the transgenic lines expressing the CAHB12 gene exhibited higher fresh weight measures than those observed for wild plants germinated in a culture medium containing 100 mM of NaCl. Besides, the level of lipid peroxidation remained virtually unchanged in two of the three transgenic lines tested, when cultivated in the presence of 100 mM of NaCl. In this context, the present invention may be used to impart greater tolerance to drought and salt stress of plants, further obtaining better performance with regard to salt stress than that observed and described in patent WO 04/099365.
The sequences with access number GT13253 and GT015128 were deposited at the public databank GenBank (National Center for Biotechnology Information), on Sep. 1, 2009, exhibiting, respectively, 89 and 92% identity with the CAHB12 gene. Although the article in which the sequences GT13253 and GT015128 are listed suggests the expression of these sequences in conditions of biotic and abiotic stress, this article has not been published yet, and to there is no proven experimental evidence, be it the expression of these transcripts in such situations or a greater tolerance of transgenic plants to water deficit and salt stress, when overexpressing such transcripts. In addition, there is no direct correlation between the above-cited sequences and the CAHB12 gene. The fact that these genes belong to the HD-Zip family alone is not sufficient to prove their efficiency in generating more tolerance phenotypes, as for example the ATHB-7 and ATHB-12 genes, which, in spite of not exhibiting an increased expression ion situations of water deficit, do not lead to a phenotype of greater resistance with respect to transgenic plants overexpressing these genes.